Eosinophilic esophagitis (EoE) is a chronic, immune-mediated, allergic disease characterized by remarkable eosinophil accumulation in the esophageal mucosa. Single-cell RNA sequencing (scRNA-Seq) indicated that the pathogenic tissue Th2 cells are specifically enriched in EoE tissue, with a large amount of Th2 cytokine production and a unique signature. Notably, by bulk and single-cell CD3+ T cell sequencing, we identified that the gene of the short-chain fatty acid (SCFA, e.g., butyrate) receptor FFAR3 is the top tracking gene with IL5, with both genes uniquely expressed in tissue Th2 cells at the single-cell level. FFAR3 can be induce by IL-4 in circulating nave T cells. We also found that butyrate, a putative ligand of FFAR3 and a metabolite produced by esophageal microbiota, significantly enhanced Th2 cytokine production in a human and mouse cell line, primary T cells, and a model of murine experimental asthma. These collective findings prompt our central hypothesis Our overall hypothesis is that FFAR3, ligated to microbiome-originated SCFA in esophageal mucosa, is induced by IL-4 in tissue effector Th2 cells and enhances type 2 responses in esophageal T cells that locally contribute to EoE pathogenesis. The proposed study will delineate unique populations of tissue Th2 cells and elucidate a novel and pivotal role of FFAR3-SCFA axis in Th2 activation, Th2 cytokine production and EoE molecular pathogenesis. Our preliminary data show that FFAR3 is an IL-4?induced SCFA receptor that regulates Th2 cytokine production by Th2 cells in vivo and ex vivo. We will substantiate whether the boosting effect of butyrate in circulating T cells occurs in tissue-resident T cells and whether there is a difference between normal control and EoE; measure the SCFA concentrations in the EoE and control esophageal tissue; and assess the SCFA and dietary fiber content in an EoE vs. non-EoE diet. We will also employ a T cell?specific FFAR3-deficient mouse to study the involvement of SCFA in murine Th2 immunity. By ChIP-Seq and ATAC-Seq, we will define epigenetic alterations induced by SCFAs, explore FFAR3's roles in altering chromatin structure at the Th2 cytokine locus, identify new Th2-promoting transcription factors driven by FFAR3 and analyze binding of the Th2 transcription factor GATA3 to genomic DNA regions in human T cells in the context of butyrate exposure. To profile the FFAR3+ tissue Th2 cells with high resolution, we will characterize a cohort of 5000 residential esophageal lymphocytes by scRNA-Seq, analyze Th2-specific markers and transcription factors, scrutinize the TCR clonotypes of tissue Th2 cells and correlate tissue Th2 cells' key parameters to EoE clinical characteristics. Collectively, this grant focuses on understanding human tissue-resident T cells and their contribution to EoE in the context of FFAR3-SCFA axis. The immediate significance of this study is its potential to uncover tissue Th2 cytokine production mechanisms, the novel functionality of SCFA in Th2 inflammation and the characterization of the newly identified FFAR3+ tissue Th2 cells, all of which may provide new rationales for improved therapeutic strategies for allergic disorders.